Methods for detecting membrane derived caspase activity and modulators thereof

ABSTRACT

Provided are methods for detecting membrane derived apoptotic activity. In one embodiment, the present invention provides methods for identifying membrane derived caspase activity. In other embodiments, drug discovery methods are provided for screening compounds that inhibit or enhance membrane derived caspase activity. In the various embodiments, heavy membrane fractions are utilized for the screening methodologies described herein.

TECHNICAL FIELD

[0001] The present invention relates generally to methods for detectingmembrane derived caspase activity and modulators thereof, and moreparticularly to novel cell-free screening assays for identifyinginhibitors and enhancers of membrane derived caspase activity.

BACKGROUND OF THE INVENTION

[0002] Tissue homeostasis is maintained by the process of apoptosis-thatis, the normal physiological process of programmed cell death. Changesto the apoptotic pathway that prevent or delay normal cell turnover areoften as important in the pathogenesis of diseases as are abnormalitiesin the regulation of the cell cycle. Like cell division, which iscontrolled through complex interactions between cell cycle regulatoryproteins, apoptosis is similarly regulated under normal circumstances bythe interaction of gene products that either function to prevent orinduce cell death.

[0003] Since apoptosis functions in maintaining tissue homeostasis in arange of physiological processes, such as embryonic development, immunecell regulation and normal cellular turnover, the dysfunction or loss ofregulated apoptosis can lead to a variety of pathological diseasestates. For example, the loss of apoptosis can lead to the accumulationof self-reactive lymphocytes associated with many autoimmune diseases.Inappropriate loss or inhibition of apoptosis can also lead to theaccumulation of virally infected cells and hyperproliferative cells,such as neoplastic or tumor cells. Similarly, the inappropriateactivation of apoptosis can contribute to a variety of pathologicaldisease states including, for example, acquired immunodeficiencysyndrome (AIDS), neurodegenerative diseases and ischemic injury.

[0004] Although apoptosis is mediated by diverse signals and complexinteractions of cellular gene products, the results of theseinteractions ultimately feed into a cell death pathway that isevolutionarily conserved between humans and invertebrates. The pathway,itself, is a cascade of proteolytic events analogous to that of theblood coagulation cascade.

[0005] Several gene families and products that modulate the apoptoticprocess have now been identified. One family is the aspartate-specificcysteine proteases (“caspases”). The caspase Ced-3, identified in C.elegans, is required for programmed cell death during development of theroundworm C. elegans. Ced-3 homologues as well as other caspases havebeen characterized. The human caspase family includes, for example,Ced-3, human ICE (interleukin-1-β converting enzyme) (caspase-1), ICH-1(caspase-2), CPP32 (caspase-3), ICE_(rel)II (caspase-4), ICE_(rel)III(caspase-5), Mch2 (caspase-6), ICE-LAP3 (caspase-7), Mch5 (caspase-8),ICE-LAP6 (caspase-9), Mch4 (caspase-10), caspase-11, caspase-12,caspase-13, caspase-14, and others.

[0006] The caspase family of cysteine proteases are essential effectorsof the apoptotic process (Yuan et al., Cell 75:641-652, 1993; Alnemri etal., Cell 87:171, 1996; Cohen, Biochem. 326:1-16, 1997; Miller, Semin.Immunol 9:3549, 1997; Salvesen and Dixit, Cell 91:443-446, 1997).Caspases are synthesized as inactive zymogens, which are activated byproteolytic processing to yield large (˜18 kDa) and small (˜12 kDa)subunits that associate to form active enzymes (Thornberry et al.,Nature 396:768-774, 1992; Nicholson et al., Nature 376:37-43, 1995;Stennicke and Salvesen, J. Biol. Chem. 272:25719-25723, 1997). Diverseapoptotic stimuli cause the activation of specific caspases which theninitiate a protease cascade by proteolytically processing additionalcaspases (Srinivasula et al., Proc. Natl. Acad. Sci. USA 93:14486-14491,1996; Yu et al., Cancer Res. 58:402-408, 1998). Once activated, thesedownstream (executioner) caspases kill cells by cleaving specificmolecular targets that are essential for cell viability or by activatingpro-apoptotic factors (Liu et al., Cell 89:175-184, 1997; Enari et al.,Nature 391:43-50, 1998; Salvesen and Dixit, Cell 91:443-446, 1997).Although caspases have been generally shown to be cytosolic proteins(Miller et al., J. Biol. Chem. 268:18062-18069, 1993; Nicholson et al.,Nature 376:37-43, 1995; Li et al., J. Biol. Chem. 272:30299-30305,1997), immunochemical studies have suggested that in some instances,caspases might also be associated with the nucleus or plasma membrane(Singer et al., J. Exp. Med. 182:1447-1459, 1995; Krajewski et al.,Blood 89:3817-3825, 1997; Posmantur et al., J. Neurochem. 68:2328-2337,1997). Recently published data has also indicated an association ofcertain caspases with mitochondria and endoplasmic reticulum (Mancini etal., J. Cell Biol. 140:1485-1495, 1998; Chandler etal., J. Biol. Chem.273:10815-10818, 1998).

[0007] The Bcl-2 family constitutes another key set of regulators of theapoptotic pathway. These proteins can function to modulate apoptosis ina wide variety of cell systems (Oltvai and Korsmeyer, Cell 79:189-192,1994; Reed, Nature 387:773-776, 1997). Bcl-2 family proteins contain oneto four conserved domains, designated BH1-BH4, and most family memberscontain a carboxyl-terminal transmembrane anchor sequence which allowsthem to be associated with cellular membranes including the outermembrane of the mitochondria, the nuclear envelope and the endoplasmicreticulum (Reed, Nature 387:773-776, 1997; Krajewski et al., Cancer Res.53:4701-4714, 1993; Yang et al., J. Cell. Biol. 128:1173-1184, 1995;Lithgow et al., Cell Growth Differ 3:411-417, 1994). The over-expressionof Bcl-2 has been shown to inhibit the activation of cytoplasmiccaspases following apoptoic stimuli in several cell systems (Armstronget al., J. Biol. Chem. 271:16850-16855, 1996; Chinnaiyan et al., J.Biol. Chem. 271:4573-4576, 1996; Boulakia et al., Oncogene 12:29-36,1996; Srinivasan et al., J. Neurosci. 16:5654-60, 1996). Moreover,previous work has demonstrated that Bcl-2 inhibits the onset ofapoptosis, but once apoptosis is initiated, Bcl-2 does not impede theprocess (McCarthy et al., J. Cell Biol. 136:215-217, 1997). However, itremains unclear how the membrane bound Bcl-2 exerts control over thesoluble cytoplasmic caspases. Further, no suitable methods exist forstudying membrane bound Bcl-2 and its effects on caspase activity in acell free manner.

[0008] The identification of compounds that modulate the apoptoticpathway via enhancement or inhibition of membrane derived caspaseactivity has been hindered by the lack of such methods. Availablemethods are limited by the lack of specificity, efficiency, and/orutilization of whole cells or cytoplasmic extracts thereof. For example,most anti-cancer drugs are screened for their ability to kill cells andtherefore will identify compounds that induce both necrosis orapoptosis. In addition, many other assay techniques focus on studyingthe inhibition or enhancement of caspase enzymes located further intothe cascade. Therefore, there exists a need in the art for methods ofidentifying compounds that not only inhibit or enhance cell death, butalso compounds that modulate the initiation of the apoptotic cascade.The present invention fulfills this need, while further providing otherrelated advantages.

[0009] The foregoing characteristics, and others which shall bedescribed in greater detail below, make the methodologies describedherein particularly attractive for drug discovery applications.

SUMMARY OF THE INVENTION

[0010] The present invention generally provides methods for detectingmembrane derived caspase activity and methods for identifying modulatorsthereof. In one aspect, the invention provides a method for identifyingmembrane derived caspase activity, that includes, incubating a membranefraction comprising heavy or nuclear membranes under conditions and fora time sufficient to allow for the evolution of caspase activity, andsubsequently detecting caspase activity.

[0011] In another aspect, the present invention provides a method foridentifying an inhibitor of the activity of a membrane derived caspase,that includes, contacting a membrane fraction with a caspase substratein the presence and absence of at least one candidate inhibitor; andcomparing the levels of caspase substrate turnover, and therefromidentifying an inhibitor of the activity of a membrane derived caspase.

[0012] In yet another aspect, the present invention provides a methodfor identifying an enhancer of the activity of a membrane derivedcaspase, that includes, contacting a membrane fraction with a caspasesubstrate in the presence and absence of at least one candidateenhancer; and comparing the levels of caspase substrate turnover, andtherefrom identifying an enhancer of the activity of a membrane derivedcaspase.

[0013] A further aspect of the present invention is a method foridentifying an inhibitor or enhancer of the evolution of caspaseprocessing within a membrane fraction, that includes, contacting amembrane fraction with at least one candidate inhibitor or candidateenhancer; and detecting the presence of large and small caspasesubunits, and therefrom determining the level of caspase processing,wherein a decrease in processing indicates the presence of a caspaseprocessing inhibitor, and wherein an increase in processing indicatesthe presence of a caspase processing enhancer.

[0014] In other embodiments, the present invention provides a method ofidentifying a compound that modulates membrane fraction derived caspaseactivity, that includes, incubating a membrane fraction, an inhibitor ofapoptosis, and a caspase substrate in the presence and absence of atleast one candidate compound under conditions and for a time sufficientto allow for the evolution of caspase activity; and comparing the levelsof caspase substrate turnover, thereby identifying a compound thatmodulates membrane derived caspase activity.

[0015] In other embodiments, inhibitors and enhancers of the activity ofa membrane derived caspase that are identified by the various methodsare provided.

[0016] In the various embodiments, caspase activity is detected bymeasuring substrate turnover or caspase processing. In otherembodiments, substrate turnover is measured by time course or endpointanalysis. In further embodiments, the membrane fraction comprises heavyor nuclear membranes. In yet further embodiments, the membrane fractionis derived from cells expressing an anti-apoptotic polypeptide. In evenfurther embodiments, the membrane fraction is derived from non-apoptoticcells.

[0017] These and other aspects of the present invention will becomeevident upon reference to the following detailed description andattached drawings. In addition, the various references set forth belowthat describe in more detail certain procedures or compositions (e.g.,plasmids, etc.), and are therefore incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 is a scanned image of an in immunoblot representingSDS-PAGE analysis of subcellular fractions from 697-neo and 697-Bcl-2cells using antibodies to PARP, cytochrome oxidase (subunit IV), D4GDIand Bcl-2. Nuc=nuclear fraction, HM=heavy membrane fraction, LM=lightmembrane fraction, S100=cytosolic fraction. Arrows indicate the specificimmunoreactive band.

[0019] FIGS. 2A-D are histograms of caspase substrate cleavage activityin subcellular fractions.

[0020]FIGS. 3A and 3B are graphs representing membrane-associatedprocaspase-3 spontaneous activation. FIG. 3A illustrates the spontaneousactivation of caspase activity in heavy membrane from 697-neo and697-Bcl-2 cells as a function of DEVD-amc turnover. FIG. 3B illustratesthe generation of soluble caspase activity from membranes as a functionof DEVD-amc turnover.

[0021]FIG. 4 is a scanned image of an immunoblot representing SDS-PAGEanalysis of heavy membrane and cytosolic fractions from 697-neo and697-Bcl-2 cells, probed with an anti-caspase-3 polyclonal antibody. Thearrowheads indicate the migration of protein size markers (RainbowMarkers, Novex); the arrow indicates procaspase-3. HM=heavy membranefractions; S100=cytosolic fraction.

[0022]FIG. 5 is a graph illustrating activation of membrane associatedDEVD-amc cleavage activity by exogenous caspase-1.

[0023]FIGS. 6A and 6B are graphical representations of DEVD-amc cleavageactivity in 697-neo and 697-Bcl-2 cells in the presence and absence ofcytochrome c. FIG. 6A illustrates the caspase activity present in theheavy membrane fraction. FIG. 6B illustrates the caspase activitypresent in the cytoplasmic fraction.

[0024]FIGS. 7A, 7B, and 7C are graphs representing the effects ofpermeabilizing detergents on membrane-associated caspase activity. FIG.7A is a graph demonstrating the effects of NP40 on spontaneous andinduced caspase activities in neo-membranes. FIG. 7B is a graphillustrating the effect of NP-40 on spontaneous caspase activation inBcl-2 and neo-membranes. FIG. 7C is a graph depicting NP-40-dependentand independent activation of procaspase-3 by granzyme B treatment ofmitochondrial enriched fractions.

DETAILED DESCRIPTION OF THE INVENTION

[0025] As noted above, the present invention is generally directed tomethods of detecting and modulating membrane derived caspase activity.One application of the disclosed invention is in the identification ofinhibitors or enhancers of apoptosis. In simple terms, the use of such anovel cell-free assay system provides a means for identifying compoundswhich promote or inhibit programmed cell death at a critical initiationpoint (i.e., membranes). Another aspect of the subject invention is theability of the disclosed assay system to investigate the effects ofmembranes derived from cells over-expressing apoptotic pathway proteins,such as those of the bcl-2 family.

[0026] As described herein, a preferred assay system utilizes heavy ornuclear membranes for detecting membrane derived caspase activity and/orfor identifying compounds that modulate caspase activity, directly orindirectly, in a cell-free system. Therefore, by using such membranesystems, control points upstream of the cytoplasmic apoptotic pathwaycan be effectively assayed and modulators thereof may be identified.

[0027] The assay methods of the present invention are particularlyuseful for drug discovery, in part by use of high throughputmethodologies. Accordingly, by utilizing the cell-free assay system ofthe present invention, identification of compounds that affect evolutionof caspase activity from the membrane fraction is rapidly achieved.

[0028] Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms thatwill be used hereinafter.

[0029] As used herein, a “caspase” refers to a cysteine protease thatspecifically cleaves proteins after Asp residues. Caspases are initiallyexpressed as zymogens, in which a large subunit is N-terminal to a smallsubunit. Caspases are generally activated by cleavage at internal Aspresidues. These proteins have been identified in many eukaryotes,including C. elegans, Drosophila, mouse, and human. Currently, there areat least 14 known caspase genes, named caspase-1 through caspase-14.Table 1 recites ten human caspases along with their alternative names.Caspase Alternative name Caspase-1 ICE Caspase-2 ICH-1 Caspase-3 CPP32,Yama, apopain Caspase-4 ICE_(rel)II; TX, ICH-2 Caspase-5 ICE_(rel)III;TY Caspase-6 Mch2 Caspase-7 Mch3, ICE-LAP3, CMH-1 Caspase-8 FLICE; MACH;Mch5 Caspase-9 ICE-LAP6; Mch6 Caspase-10 Mch4, FLICE-2

[0030] Within the context of this invention, it should be understoodthat a caspase includes wild-type protein sequences, as well as othervariants (including alleles) of the native protein sequence. Briefly,such variants may result from natural polymorphisms or may besynthesized by recombinant methodology, and differ from wild-typeprotein by one or more amino acid substitutions, insertions, deletions,or the like. Typically, when engineered, amino acid substitutions willbe conservative, i.e., substitution of amino acids within groups ofpolar, non-polar, aromatic, charged, etc. amino acids. In the region ofhomology to the native sequence, variants should preferably have atleast 90% amino acid sequence identity, and within certain embodiments,greater than 92%, 95%, or 97% identity. Such amino acid sequenceidentity may be determined by standard methodologies, including use ofthe National Center for Biotechnology Information BLAST searchmethodology available at www.ncbi.nlm.nih.gov. The identitymethodologies preferred are those described in U.S. Pat. No. 5,691,179and Altschul etal., Nucleic Acids Res. 25:3389-3402, 1997 all of whichare incorporated herein by reference. If Gapped BLAST 2.0 is utilized,then it is utilized with default settings.

[0031] As will be appreciated by those skilled in the art, a nucleotidesequence encoding a caspase or variant may differ from the known nativesequences, due to codon degeneracies, nucleotide polymorphisms, or aminoacid differences. In other embodiments, variants should preferablyhybridize to the native nucleotide sequence at conditions of normalstringency, which is approximately 25-30° C. below Tm of the nativeduplex (e.g, 5×SSPE, 0.5% SDS, 5×Denhardt's solution, 50% formamide, at42° C. or equivalent conditions; see generally, Sambrook et al.Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring HarborPress, 1987; Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing, 1987).

[0032] An “isolated nucleic acid molecule” refers to a polynucleotidemolecule in the form of a separate fragment or as a component of alarger nucleic acid construct, that has been separated from its sourcecell (including the chromosome it normally resides in) at least once ina substantially pure form. Nucleic acid molecules may be comprised of awide variety of nucleotides, including DNA, RNA, nucleotide analogues,or some combination of these.

[0033] A “membrane fraction”, as used herein, refers to a subcellularfraction of a eukaryotic cell comprising cellular membranes. Inparticular, the term “heavy membranes”, as used herein, refers to asubcellular fraction substantially tree or nuclear and light membranes,wherein predominant components is mitochondria.

[0034] A “stimulator of apoptosis” or “pro-apoptotic agent”, as usedherein refers to an agent that increases the specific apoptotic activityof a cell. Illustrative examples of such stimulus are deprivation of agrowth factor, Fas ligand, anti-Fas antibody, staurosporine, ultravioletirradiation, gamma irradiation, tumor necrosis factor, and others wellknown in the art. Accordingly, a stimulator of apoptosis is an agentthat increases the molecular activity of caspase molecules eitherdirectly or indirectly. In addition, a stimulator of apoptosis can be apolypeptide that is capable of increasing or inducing the apoptoticactivity of a cell. Such polypeptides include those that directlyregulate the apoptotic pathway such as Bax, Bad, Bcl-xS, Bak, Bik, andactive caspases as well as those that indirectly regulate the pathway.

[0035] An “inhibitor of apoptosis” or “anti-apoptotic agent”, as usedherein refers to an agent that decreases the apoptotic activity of acell when compared to control agents. Illustrative examples of suchanti-apoptotic agents include small molecules, fmk p35, crma, Bcl-2,Bcl-X_(L), Mcl-1, E1B-19K from adenovirus, as well as antagonists ofpro-apoptotic agents (e.g. antisense, ribozymes, antibodies, etc.).Accordingly, an inhibitor of apoptosis is an agent that decreases themolecular activity of caspase molecules either directly or indirectly.

[0036] An “apoptotic pathway protein”, as used herein refers to aprotein involved in the cell death pathway. Illustrative examplesinclude Bcl-2, Bcl-X_(s), Bcl-X_(L), Bik, Bak, Bax, Bad, caspasemolecules, Apaf-1, cytochrome c, and the like. “Evolution of caspaseactivity”, as used herein, refers to the increasing of detectable levelsof caspase protease activity over a time period. Such evolution may beevidenced by detectable increases in substrate turnover (e.g.,fluorogenic substrates) and/or detectable increases in caspaseprocessing. “Membrane derived caspase activity”, as used herein, refersto caspase activity that is released from or associated with heavy ornuclear membranes.

[0037] A. Membrane Preparations

[0038] Membrane preparations within the context of the present inventionmay be derived from a variety of cell types or sources. Typically, forease of handling, the cells utilized will be a eukaryotic cell line orother culturable cell type. However, cells can also be derived fromtissues and other non-cultured sources. One of ordinary skill in the artwould readily appreciate that the assays of the present invention arenot dependent upon the exact source or type of cell from which membranefractions are prepared.

[0039] Subcellular fractionation has been a basic research tool in cellbiology for the last 30 years. Accordingly, those of ordinary skill inthe art are familiar with various techniques for such fractionation.Typically, subcellular fractionation comprises two basic steps, 1)homogenization and 2) separation. Homogenization in its ideal formallows particulate organelles such as the nucleus, mitochondria,lysosomes, and peroxisomes to remain intact. A variety of homogenizationtechniques are known, such as Dounce homogenizers (glass/glass),Potter-Elvehjem homogenizers (glass/teflon), repeated pipetting, passagethrough small gauge needle, and the like. Exemplary techniques aredescribed in detail by Harms et al., Proc. Natl. Acad. Sci. USA77:6139-6143 1980, Darte et al., J. Exp. Med. 157:1208-1228, 1983, andBalch et al., Cell 39:405-416, 1984.

[0040] Separation of subcellular fractions is traditionally performedusing density gradients. While sucrose gradients are the most widelyused, many other alternatives are available (e.g., Ficoll, Percoll,Metrizamide, and Nycodenz) (see Methods in Enzymology Vol. 31, Part A(Flescher and Packer eds.), 1974). In addition, a number of alternativemethods have been developed for isolation of various components,including density modification, free flow electrophoresis, andimmuno-isolation (see Cell free Analysis of Membrane Traffic, pp.35-127, (Morre et al. eds.)(1988)). Moreover, a variety of referencesare available which detail a multitude of fractionation techniques, forexample, see Methods in Enzymology Vol. 31, Part A (Flescher and Packereds.), 1974; Partition of Cell Particles and Macromolecules: Separationand purification of Biomolecules, Cell Organelles, Membranes, and Cells(Albertsson, ed.), 1986; Martin et al., Eur. J. Clin. Inv. 13:49-56,1983.

[0041] An exemplary method of cellular fractionation comprisessuspending cells in a hypotonic buffer in which a variety of proteaseinhibitors are present (e.g., PMSF, leupeptin, pepstatin, aprotinin,EDTA, etc.). The samples are incubated on ice, then homogenized using aDounce homogenizer. Following homogenization the homogenate iscentrifuged at 500×g to separate nuclei. The nuclear pellet can then bewashed and resuspended. The supernatant is then centrifuged at 14,000×gfor 30 minutes to pellet the heavy membranes. The 14,000×g supernatantcan then be centrifuged at 100,000×g for 30 minutes to yield asupernatant (cytoplasmic fraction) and a pellet (light membranefraction). The pelleted fractions can then be washed and resuspended inthe appropriate buffer for assaying.

[0042] B. Screening of Inhibitors and Enhancers of the Evolution ofCapase Activity from a Membrane Fraction

[0043] 1. Inhibitors and Enhancers of Membrane Derived Caspase Activity

[0044] Candidate inhibitors and enhancers may be isolated or procuredfrom a variety of sources, such as bacteria, fungi, plants, parasites,libraries of chemicals, peptides or peptide derivatives and the like.Inhibitors and enhancers may be also be rationally designed, based onthe protein structure determined from X-ray crystallography (see, Mittlet al., J. Biol. Chem., 272:6539-6547, 1997). In certain embodiments,the inhibitor targets a specific caspase (e.g., membrane associatedcaspases). In other embodiments, the candidate inhibitor or enhancerindirectly affects the release/evolution of membrane derived caspaseactivity.

[0045] Without being held to a particular mechanism, the inhibitor mayact by preventing processing of a caspase, preventing caspase enzymaticactivity, by other mechanisms, or by preventing liberation of thecaspase from the membrane. Accordingly, the inhibitor may act directlyor indirectly. In one embodiment, inhibitors interfere in the processingof the caspase protein. In other embodiments, the inhibitors are smallmolecules. In yet another embodiment, inhibitors interact with Bcl-2. Inother aspects, the inhibitors prevent apoptosis. Inhibitors should havea minimum of side effects and are preferably non-toxic. Inhibitors thatcan penetrate cells are preferred.

[0046] In addition, enhancers of caspase activity or expression aredesirable in certain circumstances. At times, increasing apoptosis willhave a therapeutic effect. For example, tumors or cells that mediateautoimmune diseases are appropriate cells for destruction. Enhancers mayincrease the rate or efficiency of caspase processing, increasetranscription or translation, increase caspase release/evolution fromthe membrane, or act through other mechanisms. As is apparent to oneskilled in the art, many of the guidelines presented above apply to thedesign of enhancers as well.

[0047] 2. Screening Assay Formats

[0048] Screening assays for inhibitors and enhancers will vary accordingto the type of inhibitor or enhancer and the nature of the activity thatis being affected. In general, assays, within the context of the presentinvention, are designed to evaluate caspase protein processing orcaspase enzymatic activity as the result of caspase activity thatevolves/derives from a membrane fraction. In any of the assays, astatistically significant increase or decrease compared to a propercontrol is indicative of enhancement or inhibition. Moreover, it shouldbe understood that detection of membrane derived caspase activity may beby direct or indirect means. For example, a direct means is detectingmembrane caspase substrate turnover, while an indirect means isdetecting the processing or direct activity of a caspase activated bythe membrane derived caspase.

[0049] In one embodiment, the assay utilizes membrane preparations fromeukaryotic cells. In this regard, any cell type may be used depending onthe purpose of the assay. In certain embodiments, the membrane fractioncomprises heavy membranes and/or nuclear membranes. In one aspect, themembrane fraction is contacted or contacted and incubated in thepresence or absence of a candidate inhibitor or enhancer and thesubstrate turnover or caspase-processing is measured. Substrate turnoveror caspase-processing (cleavage of caspases into large and smallsubunits) can be assessed by a variety of methods known by those ofskill in the art including, for example, fluorescence spectroscopy, massspectroscopy, HPLC, colorimetry (e.g., UV and visible spectroscopy),fluorography, radiography, gel electrophoresis,immuno-blotting/immuno-affinity, chromatography, N-terminal peptidesequencing and the like. Moreover, one of ordinary skill in the art willrecognize that incubation may be carried out at a variety oftemperatures, depending on the kinetics to be studied. In oneembodiment, the incubation temperature is from 20° C. to 40° C. In otherembodiments, the incubation temperature is from 25° C. to 37° C.

[0050] One in vitro assay can be performed by examining the effect of acandidate compound on the processing of a caspase (e.g., a pro-caspaseor other protein substrate of a caspase) into two subunits. Briefly, asubstrate (e.g., peptide, protein, or peptide mimetic) containing theenzyme recognition site of membrane derived caspase-3 is utilized (e.g.,DEVD), for example, when such a substrate is a protein or peptide, thesubstrate is in vitro translated or purified from a cell expressionsystem. This primary product is contacted or contacted and incubatedwith the membrane fraction in the presence or absence of a candidateinhibitor or enhancer and assessed for appearance of the two subunits.To facilitate detection, typically, the protein or peptide is labeledduring translation or via gene fusion prior to expression. Ifradiolabeled, the two subunits may be readily detected byautoradiography after gel electrophoresis. One skilled in the art willrecognize that other methods of labeling and detection may be usedalternatively.

[0051] An alternative in vitro assay is designed to measure cleavage ofa caspase substrate analog (e.g., Acetyl-DEVD-aminomethylcoumarin (amc),lamin, poly-(ADP-ribose)polymerase (PARP), and the like, a variety ofwhich are commercially available). Substrate turnover (e.g., substratehydrolysis) may be assayed using either comparison of timecourse (i.e.,progress curve) assays (e.g., evolution of activity and substratehydrolysis rate analysis via steady-state rate comparison) or endpointanalysis (e.g., final fluorescence minus initial fluorescence). Briefly,in this assay the membrane fraction is incubated with a candidateinhibitor or enhancer along with the caspase substrate. Detection ofcleaved substrate is performed by any one of a variety of standardmethods. Generally, the substrate will be labeled and followed by anappropriate detection means.

[0052] Typical substrates utilized within the context of the presentinvention include those agents whose turnover measures, directly orindirectly, the apoptotic pathway and, in particular, the enzymaticactivity of one or more caspase molecules. In this regard a variety ofsubstrates such as labeled caspase molecules, lamin, PARP and caspasesubstrate analogues are known by those of skill in the art. Suchsubstrates are also available commercially from such companies asOncogene Research Products, Cambridge, Mass. Illustrative substrateanalogues which are tagged with fluorescent markers include, ZEVD-amc(carbobenzoxy-Glu-Val-Asp-aminomethylcoumarin), YVAD-amc(Acetyl-Tyr-Val-Ala-Asp-aminomethylcoumarin), and DEVD-amc(Acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin).

[0053] Moreover, any known enzymatic analysis can be used to follow theinhibitory or enhancing ability of a candidate compound with regard tomembrane derived caspase activity. It would be apparent to one ofordinary skill in the art that the candidate inhibitor or enhancer maybe incubated with the cell prior to fractionation or with the membranefraction after fractionation, but prior to detection. Moreover, thecandidate inhibitor or enhancer may be contacted or contacted andincubated with the membrane fraction concurrently with a caspasesubstrate.

[0054] The assays briefly described herein may be used to identify anenhancer or inhibitor that specifically affects membrane derived caspaseactivity. A variety of methodologies exist that can be used toinvestigate the effect of a candidate compound. Such methodologies arethose commonly used to analyze enzymatic reactions and include, forexample, SDS-PAGE, spectroscopy, HPLC analysis, autoradiography,chemiluminescence, chromogenic reactions, and immunochemistry (e.g.blotting, precipitating, etc.).

[0055] Furthermore, in other assay embodiments, eukaryotic promoters maybe utilized within a construct for delivering either inducible orconstitutively expressed pro- or anti-apoptotic proteins to the cellsfrom which membrane preparations will be derived. For example, cells canbe transfected such that they overexpress the anti-apoptotic polypeptideBcl-2, thereby providing cells wherein membrane preparations would havea higher level of Bcl-2, such that only enhancers of apoptosis whichwere capable of overcoming Bcl-2 inhibition would be detected. In thissame regard, such cells could be treated with a stimulus of apoptosissuch that the cell is “poised” for apoptosis prior to subfractionation.In such a method, treatment of the membrane fraction with an apoptoticpathway enhancer results in significantly more robust activation ratethan a comparable enhancer effect on non-poised cells.

[0056] In further embodiments, cells “poised” for cell death by deliveryof an apoptotic stimulator prior to subfractionation, may be created bytreating cells that do not overexpress anti-apoptotic polypeptides, butwhich are fractionated prior to apoptosis. Such cells may besubfractionated and the membranes derived therefrom utilized forassaying candidate inhibitors and enhancers.

[0057] The methods described above for identification of inhibitors andenhancers of apoptosis provides an alternative format for measuringapoptotic activity, in that a cell is treated so that it is “poised” forprogrammed cell death. In this way the cell has synthesized and/oractivated all necessary components that are required for programmed celldeath. All that is required is a stimulus to cause the cell to extendpast its holding point and into apoptosis. Accordingly, an enhancerwould cause the cell to progress into programmed cell death, while aninhibitor would delay or suppress this progress in the presence of anapoptotic stimulus.

[0058] The holding point which prevents the cell from proceeding intoprogrammed cell death can be the overexpression of a cell survivalpolypeptide or treatment of the cells with known apoptotic inhibitors.Cell survival polypeptides are characterized in that they exhibit theability to prevent apoptosis when expressed or activated in a cellinduced to undergo apoptosis. For example, in the absence of afunctioning cell survival polypeptide, a cell treated with an apoptoticenhancer (e.g., a pro-apoptotic agent) will initiate or accelerateapoptosis. However, in the presence of a cell survival polypeptide,treatment with a pro-apoptotic agent/enhancer can initiate theprogrammed cell death pathway, but the cell will survive due toinhibition of one or more events along the pathway. Depending upon thepoint at which the cell survival polypeptide functions, the programmedcell death pathway can be inhibited early or relatively late within theexecution of the cascade of events leading to ultimate cell death. Cellsurvival polypeptides and their encoding nucleic acids are well known inthe art and include, for example, the Bcl-2 family of related proteinsBcl-2, Bcl-X_(L), Mcl-1, E1B-19K as well as inhibitors of the caspaseactivity such as p35, crmA and the dominant-negative forms of thecaspases. These forms include, for example, caspase's with aninactivating mutation of the active site cysteine.

[0059] Overexpression of a cell survival polypeptide can be achievedusing, for example, recombinant methods known to those skilled in theart. Routine procedures for performing such recombinant expressionmethods are described in, for example, Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York(1992), Greene Publishing Associates and Wiley-Interscience, New York,(1995). Such methods can be used to express stably or transiently a cellsurvival polypeptide at a level which is sufficient to prevent theinduction of apoptosis. The nucleic acid molecule encoding the cellsurvival polypeptide can be encoded by, for example, a homologousnucleic acid derived from the same species or cell type, oralternatively, the nucleic acid molecule can be encoded by aheterologous nucleic acid derived from a different species or cell type.The source of the encoding nucleic acid is not important so long as theencoded cell survival polypeptide exhibits apoptosis inhibitingactivity.

[0060] A level of expression of a cell survival polypeptide which issufficient to prevent the induction of apoptosis is known to thoseskilled in the art and can also be routinely determined by those skilledin the art. Expression vectors and systems are known and commerciallyavailable which provide for recombinant polypeptide expression. It is aroutine matter for one skilled in the art to choose a vector or systemwhich will provide sufficient levels of expression in a particular hostcell. Alternatively, the expression level sufficient to prevent theinduction of apoptosis can be routinely determined by expressing thecell survival polypeptide and then measuring whether the cell survivesafter treatment with a pro-apoptotic agent.

[0061] In addition to recombinant methods of over-expressing a cellsurvival polypeptide, a cell can be used which inherently over-expressesa cell survival polypeptide. A specific example of a cell inherentlyover-expressing a cell survival polypeptide is the B cell lymphoma inwhich Bcl-2 was initially identified. This leukemia has a translocationof chromosome 14 to 18 causing high level expression of Bcl-2 andtherefore cell survival. The leukemic phenotype is due to the increasedcell survival. Other cell lines which inherently over-express a cellsurvival polypeptide can similarly be used in the methods of theinvention.

[0062] The block from apoptosis due to over-expression of a cellsurvival polypeptide and the treatment of the cells with a pro-apoptoticagent provides antagonistic influences to the cell. In this way, thecells are essentially poised for programmed cell death. A pro-apoptoticagent can be a variety of different insults to the cell including,molecular, environmental and physical stimuli. As defined previously,such stimuli are known to those skilled in the art and can becharacterized by activating a molecule within the apoptotic pathway.Examples of pro-apoptotic agents include inducers such as deprivation ofa growth factor, Fas ligand, anti-Fas antibody, staurosporine, TumorNecrosis Factor, ultraviolet and gamma-irradiation. Thus, treatment of acell over-expressing a cell survival polypeptide with a pro-apoptoticagent will prime the cell for apoptosis since both positive and negativesignals provide balancing effects. One advantage of this priming is thatall cell death components are available for apoptosis once a signal isreceived that overcomes the block of the cell survivalpolypeptide/anti-apoptotic agent. This allows for the rapid induction ofapoptosis which can be use in screening for compounds that possessapoptosis inducing activity in the presence of Bcl-2 or Bcl-X_(L). Suchcells are particularly useful in screening for inhibitors of Bcl-2 orBcl-X_(L), respectively.

[0063] 3. High throughput

[0064] The methods described herein are also amenable to high throughputformats (e.g., a multi-well format assay where large numbers of samplescan be screened rapidly and efficiently). For example, a 96-well formatprovides practical advantages since plates appropriate for manipulationsand measuring devices are commercially available. Such procedures can befurther automated to increase further the speed and efficiency of themethod. These features, combined with the specificity of the method,allow for cell-free high throughput screening of candidate inhibitors orenhancers of caspase activity derived from membranes. For example, alibrary of test compounds can be administered to a plurality of membranesamples and then assayed for their ability to enhance or inhibitapoptosis. Identified compounds are valuable for both therapeutic anddiagnostic purposes since they can allow for the treatment and detectionof apoptotic mediated diseases. Such compounds are also valuable inresearch related to apoptotic mechanisms given that they can help deducefurther molecular events and provide further specificity for thediscovery and development of future compounds.

[0065] C. Caspase and Apoptotic Pathway Genes

[0066] As noted above, the invention provides assay methods relating tocaspase and other apoptotic pathway genes and gene products, and methodsfor the use of the genes and gene products. In particular, the inventionprovides assays that detect modulation of membrane derived caspaseactivity. Given the disclosure provided herein, and the knowledge ofthose skilled in the art, an apoptotic pathway protein encoding gene canbe isolated from a variety of cell types.

[0067] 1. Isolation of Apoptotic Protein Encoding Genes

[0068] Apoptotic protein encoding genes may be isolated from eithergenomic DNA or preferably cDNA. Isolation of apoptotic pathway genesfrom genomic DNA or cDNA typically can proceed by, first, generating anappropriate DNA library through techniques for constructing librariesthat are known in the art (see Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, 1989) or purchased fromcommercial sources (e.g., Clontech, Palo Alto, Calif.). Briefly, cDNAlibraries can be constructed in bacteriophage vectors (e.g, λZAPII),plasmids, or others, which are suitable for screening, while genomic DNAlibraries can be constructed in chromosomal vectors, such as YACs (yeastartificial chromosomes), bacteriophage vectors, such as λEMBL3, λgt10,cosmids, or plasmids.

[0069] In one embodiment known apoptotic protein gene sequences(caspase, Bcl-2, Bcl-X_(s), Bcl-X_(L), Bik, Bad, Bax, etc.) may beutilized to design an oligonucleotide hybridization probe suitable forscreening genomic or cDNA libraries. Preferably, such oligonucleotideprobes are 20-30 bases in length. To facilitate hybridization detection,the oligonucleotide may be conveniently labeled, generally at the 5′end, with a reporter molecule, such as a radionuclide, (e.g., ³²P),enzymatic label, protein label, fluorescent label, or biotin. Suchlibraries are then generally plated as phage or colonies, depending uponthe vector used. Subsequently, a nitrocellulose or nylon membrane, towhich the colonies or phage have been transferred, is probed to identifycandidate clones which contain the apoptotic pathway gene. Suchcandidates may be verified as containing the target DNA by any ofvarious means including, for example, DNA sequence analysis orhybridization with a second, non-overlapping probe.

[0070] Once a library is identified as containing an apoptotic proteingene, the gene can be isolated by amplification. Briefly, using acaspase gene as an illustration, when using cDNA library DNA as atemplate amplification primers are designed based upon known caspasegene sequences (see GenBank Accession Nos. X65019 (caspase-1), U13021(caspase-2), U13737 (caspase-3), U25804 (caspase-4), U28015 (caspase-5),U20536 (caspase-6), U37448 (caspase-7), U60520 (caspase-8), U56390(caspase-9), U60519 (caspase-10), and sequences available in the art).Amplification of cDNA libraries made from cells with high caspaseactivity is preferred. Primers for amplification are preferably derivedfrom sequences in the 5′ and 3′ untranslated region in order to isolatea full-length cDNA. The primers preferably have a GC content of about50% and contain restriction sites to facilitate cloning and do not haveself-complementary sequences nor do they contain complementary sequencesat their 3′ end (to prevent primer-dimer formation). The primers areannealed to cDNA or genomic DNA and sufficient amplification cycles areperformed to yield a product readily visualized by gel electrophoresisand staining. The amplified fragment is purified and inserted into avector, such as λgt10 or pBS(M13+), and propagated. Confirmation of thenature of the fragment is obtained by DNA sequence analysis orindirectly through amino acid sequencing of the encoded protein.

[0071] Other methods may also be used to obtain the apoptotic pathwayprotein encoding nucleic acid molecule. For example, a nucleic acidmolecule encoding caspase may be obtained from an expression library byscreening with an antibody or antibodies reactive to caspase (see,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., ColdSpring Harbor Laboratory Press, NY, 1987; Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates andWiley-Interscience, NY, 1995).

[0072] Variants of apoptotic pathway protein genes may be engineeredfrom natural variants (e.g., polymorphisms, splice variants, mutants),synthesized or constructed. Many methods have been developed forgenerating mutants (see, generally, Sambrook et al., supra; Ausubel, etal., supra, and the discussion above). Briefly, preferred methods forgenerating a few nucleotide substitutions utilize an oligonucleotidethat spans the base or bases to be mutated and contains the mutated baseor bases. The oligonucleotide is hybridized to complementary singlestranded nucleic acid and second strand synthesis is primed from theoligonucleotide. The double-stranded nucleic acid is prepared fortransformation into host cells, typically E. coli, but alternatively,other prokaryotes, yeast or other eukaryotes. Standard screening andvector growth protocols are used to identify mutant sequences and obtainhigh yields.

[0073] Similarly, deletions and/or insertions of genes may beconstructed by any of a variety of known methods as discussed supra. Forexample, the gene can be digested with restriction enzymes and religatedsuch that a sequence is deleted or religated with additional sequencessuch that an insertion or large substitution is made. Other means ofgenerating variant sequences may be employed with methods known in theart, for example those described in Sambrook et al. (supra) and Ausubelet al. (supra). Verification of variant sequences is typicallyaccomplished by restriction enzyme mapping, sequence analysis, or probehybridization.

[0074] D. Vectors, Host Cells and Means of Expressing and ProducingProtein

[0075] An apoptotic pathway protein may be expressed in a variety ofhost organisms. In certain embodiments, the protein is produced inbacteria, such as E. coli, or mammalian cells (e.g., CHO and COS-7), forwhich many expression vectors have been developed and are available.Other suitable host organisms include other bacterial species, andeukaryotes, such as yeast (e.g., Saccharomyces cerevisiae), and insectcells (e.g., Sf9).

[0076] A DNA sequence encoding the protein is introduced into anexpression vector appropriate for the host. In certain embodiments, theprotein can be is inserted into a vector such that a fusion protein isproduced. As discussed above, the sequence may contain alternativecodons for each amino acid with multiple codons. The alternative codonscan be chosen as “optimal” for the host species. Restriction sites aretypically incorporated into the primer sequences and are chosen withregard to the cloning site of the vector. If necessary, translationalinitiation and termination codons can be engineered into the primersequences.

[0077] At a minimum, the vector must contain a promoter sequence. Asused herein, a “promoter” refers to a nucleotide sequence that containselements that direct the transcription of a linked gene and contains anRNA polymerase binding site. More typically, in eukaryotes, promotersequences contain binding sites for other transcriptional factors thatcontrol the rate and timing of gene expression. Such sites include TATAbox, CAAT box, POU box, AP1 binding site, and the like. Promoter regionsmay also contain enhancer elements. When a promoter is linked to a geneso as to enable transcription of the gene, it is “operatively linked.”

[0078] Other regulatory sequences may be included. Such sequencesinclude a transcription termination signal sequence, secretion signalsequence, origin of replication, selectable marker, and the like. Theregulatory sequences are operationally associated with one another toallow transcription or translation.

[0079] The expression vectors used herein include a promoter designedfor expression of the proteins in a host cell (e.g., bacterial).Suitable promoters are widely available and are well known in the art.Inducible or constitutive promoters are preferred. Such promoters forexpression in bacteria include promoters from the T7 phage and otherphages, such as T3, T5, and SP6, and the trp, lpp, and lac operons.Hybrid promoters (see, U.S. Pat. No. 4,551,433), such as lacVV, tac, andtrc, may also be used. Promoters for expression in eukaryotic cellsinclude the P10 or polyhedron gene promoter of baculovirus/insect cellexpression systems (see, e.g., U.S. Pat. Nos. 5,243,041, 5,242,687,5,266,317, 4,745,051, and 5,169,784), MMTV LTR, CMV IE promoter, RSVLTR, SV40, metallothionein promoter (see, e.g., U.S. Pat. No. 4,870,009)and the like.

[0080] The promoter controlling transcription of the apoptotic pathwayprotein may itself be controlled by a repressor. In some systems, thepromoter can be derepressed by altering the physiological conditions ofthe cell, for example, by the addition of a molecule that competitivelybinds the repressor, or by altering the temperature of the growth media.Preferred repressor proteins include, but are not limited to the E. colilacI repressor responsive to IPTG induction, the temperature sensitiveλcI857 repressor, and the like.

[0081] In other preferred embodiments, the vector also includes atranscription terminator sequence. A “transcription terminator region”has either a sequence that provides a signal that terminatestranscription by the polymerase that recognizes the selected promoterand/or a signal sequence for polyadenylation.

[0082] Preferably, the vector is capable of replication in the hostcells. Thus, when the host cell is a bacterium, the vector preferablycontains a bacterial origin of replication. Preferred bacterial originsof replication include the p15A, pSC101, and col E1 origins ofreplication, especially the ori derived from pUC plasmids. In yeast, ARSor CEN sequences can be used to assure replication. A well-used systemin mammalian cells is SV40 ori.

[0083] The plasmids also preferably include at least one selectablemarker that is functional in the host. A selectable marker gene includesany gene that confers a phenotype on the host that allows transformedcells to be identified and selectively grown. Suitable selectable markergenes for bacterial hosts include the ampicillin resistance gene(Amp^(r)), tetracycline resistance gene (Tc^(r)) and the kanamycinresistance gene (Kan^(r)). The kanamycin resistance gene is presentlypreferred. Suitable markers for eukaryotes usually require acomplementary deficiency in the host (e.g., thymidine kinase (tk) in tk−hosts). However, drug markers are also available (e.g., G418 resistanceand hygromycin resistance).

[0084] One skilled in the art appreciates that there are a wide varietyof suitable vectors for expression in bacterial cells and which arereadily obtainable. Vectors such as the pET series (Novagen, Madison,Wis.), the tac and trc series (Pharmacia, Uppsala, Sweden), pTTQ18(Amersham International plc, England), pACYC 177, pGEX series, and thelike are suitable for expression of the protein. Baculovirus vectors,such as pBlueBac (see, e.g., U.S. Pat. Nos. 5,278,050, 5,244,805,5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784; availablefrom Invitrogen, San Diego) may be used for expression in insect cells,such as Spodoptera frugiperda sf9 cells (see, U.S. Pat. No. 4,745,051).The choice of a bacterial host for the expression of an apoptoticpathway protein is dictated in part by the vector. Commerciallyavailable vectors are paired with suitable hosts.

[0085] A wide variety of suitable vectors for expression in eukaryoticcells are available. Such vectors include pCMVLacI, pXT1 (StratageneCloning Systems, La Jolla, Calif.); pCDNA series, pREP series, pEBVHis(Invitrogen, Carlsbad, Calif.). In certain embodiments, the gene ofinterest is cloned into a gene targeting vector, such as pMC1neo, a pOGseries vector (Stratagene Cloning Systems).

[0086] The apoptotic pathway protein is isolated by standard methods,such as affinity chromatography, size exclusion chromatography, metalion chromatography, ionic exchange chromatography, HPLC, and other knownprotein isolation methods. (see generally Ausubel et al. supra; Sambrooket al. supra). An isolated protein gives a single band on SDS-PAGE whenstained with Coomassie blue.

[0087] Apoptotic pathway proteins may be expressed as a hexa-his (His)₆fusion protein and isolated by metal-containing chromatography, such asnickel-coupled beads. Briefly, a sequence encoding His₆ is linked to aDNA sequence encoding the desired protein. Although the His₆ sequencecan be positioned anywhere in the molecule, preferably it is linked atthe 3′ end immediately preceding the termination codon. The fusion maybe constructed by any of a variety of methods.

[0088] E. Use of Inhibitors or Enhancers

[0089] Inhibitors and enhancers may be used in the context of thisinvention to exert control over the cell death process or cytokineactivation (e.g., IL-1, which is activated by caspase-1). Thus, theseinhibitors and enhancers will have utility in diseases characterized byeither excessive or insufficient levels of apoptosis. Inhibitors ofproteases have potential to treat the major neurodegenerative diseases:stroke, Parkinson's Disease, Alzheimer's Disease, and ALS. As well,caspase protease inhibitors may be used to inhibit apoptosis in theheart following myocardial infarction, in the kidney following acuteischemia, and in diseases of the liver. Enhancers of caspase activitymay be used in contexts when apoptosis or cytokine activation aredesired. For example, inducing or increasing apoptosis in cancer cellsor aberrantly proliferating cells may be effected by delivery of acaspase enhancer.

[0090] The inhibitors and enhancers may be farther coupled with atargeting moiety that binds a cell surface receptor specific to thecells. Administration of inhibitors or enhancers will generally followestablished protocols. The compounds identified by the methods of theinstant invention may be administered either alone, or as apharmaceutical composition. Briefly, pharmaceutical compositions of thepresent invention may comprise one or more of the inhibitors orenhancers as described herein, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Such compositions may comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like, carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol, proteins,polypeptides or amino acids such as glycine, antioxidants, chelatingagents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide)and preservatives. In addition, pharmaceutical compositions of thepresent invention may also contain one or more additional activeingredients.

[0091] Compositions identified by the methods of the present inventionmay be formulated for the manner of administration indicated, includingfor example, for oral, nasal, venous, intracranial, intraperitoneal,subcutaneous, or intramuscular administration. Within other embodimentsof the invention, the compositions described herein may be administeredas part of a sustained release implant. Within yet other embodiments,compositions of the present invention may be formulized as alyophilizate, utilizing appropriate excipients which provide stabilityas a lyophilizate, and subsequent to rehydration.

[0092] As noted above, pharmaceutical compositions also are provided bythis invention. These compositions may contain any of the abovedescribed inhibitors, enhancers, DNA molecules, vectors or host cells,along with a pharmaceutically or physiologically acceptable carrier,excipients or diluents. Generally, such carriers should be nontoxic torecipients at the dosages and concentrations employed. Ordinarily, thepreparation of such compositions entails combining the therapeutic agentwith buffers, antioxidants such as ascorbic acid, low molecular weight(less than about 10 residues) polypeptides, proteins, amino acids,carbohydrates including glucose, sucrose or dextrins, chelating agentssuch as EDTA, glutathione and other stabilizers and excipients. Neutralbuffered saline or saline mixed with nonspecific serum albumin areexemplary appropriate diluents.

[0093] In addition, the pharmaceutical compositions of the presentinvention may be prepared for administration by a variety of differentroutes, including for example intraarticularly, intracranially,intradermally, intrahepatically, intramuscularly, intraocularly,intraperitoneally, intrathecally, intravenously, subcutaneously or evendirectly into a tumor. In addition, pharmaceutical compositions of thepresent invention may be placed within containers, along with packagingmaterial which provides instructions regarding the use of suchpharmaceutical compositions. Generally, such instructions will include atangible expression describing the reagent concentration, as well aswithin certain embodiments, relative amounts of excipient ingredients ordiluents (eg, water, saline or PBS) which may be necessary toreconstitute the pharmaceutical composition. Pharmaceutical compositionsare useful for both diagnostic or therapeutic purposes.

[0094] Pharmaceutical compositions of the present invention may beadministered in a manner appropriate to the disease to be treated (orprevented). The quantity and frequency of administration will bedetermined by such factors as the condition of the patient, and the typeand severity of the patients disease. Dosages may be determined mostaccurately during clinical trials. Patients may be monitored fortherapeutic effectiveness by appropriate technology, including signs ofclinical exacerbation, imaging and the like.

[0095] The following examples are offered by way of illustration, andnot by way of limitation.

EXAMPLES Example 1 Cell-Lines and Cell Culture

[0096] 697 human lymphoblastoid cells stably infected with a retroviralexpression construct containing Bcl-2 cDNA (697-Bcl-2 cells) or acontrol neomycin resistance gene (697-neo-cells) (Miyashita and Reed,1993) (obtained from Dr. John Reed, Burnham Institute) were used inthese studies. The cells were maintained in mid-log phase growth in RPMI1640 medium (Irvine Scientific, Santa Ana, Calif.) supplemented with 10% fetal bovine serum ((FBS) Hyclone, Logan, Utah), 0.2 mg/ml G-418(Gibco, Gaithersburg, Md.) and 0.1 mg/ml penicillin/streptomycin (IrvineScientific). Murine dopaminergic MN9D cells (obtained from Dr. A.Heller, University of Chicago) were grown in DMEM medium (IrvineScientific) supplemented with 10% FBS, 2 mM glutamine and 0.1 mg/mlpenicillin/streptomycin. Mouse brain cortical cells were prepared at E15of gestation in Hank's buffered saline solution (Irvine Scientific) with15 mM HEPES. The tissue was briefly dissociated with 0.1% trypsin andwashed thoroughly with MEM medium supplemented with 10% FBS and 0.4mg/ml DNase I (Sigma, St. Louis, Mo.), gently triturated and flashfrozen.

Example 2 Sub-Cellular Fractionation

[0097] Frozen cell pellets containing ≈10⁹ cells were thawed andresuspended in cold hypotonic buffer (10 mM Na-HEPES, 5 mM MgCl₂, 42 mMKCl, pH 7.4) supplemented with 1 mM PMSF, 1 μg/ml leupeptin, 1 μg/mlpepstatin A, 5 μg/ml aprotinin, 0.1 mM EDTA, 0.1 mM EGTA and 5 mM DTT(Sigma) to a density of ≈1.5×10⁸ cells/ml. The samples were incubated onice for 30 min at which time the cells were lysed using 30 -40 strokeswith a Dounce homogenizer. The sample was centrifuged twice for 10 minat 500×g, 4° C. to separate the nuclei. The nuclear pellets were thenwashed twice in the same buffer supplemented with 1.6 M sucrose,yielding the nuclear fraction. The supernatant was then centrifuged at14,000x g for 30 min at 4° C. to pellet the heavy membranes. The heavymembranes were washed 3 times with 1.5 ml cold hypotonic buffercontaining protease inhibitors and DTT. The washed membranes wereresuspended in hypotonic buffer so that the total protein concentrationwas approximately 2 mg/ml, yielding the heavy membrane fraction, thatwas either flash frozen or used immediately for enzymatic measurementswithout freezing. The 14,000×g supernatant was centrifuged at 100,000×gfor 30 min at 4° C., yielding a supernatant (cytoplasmic fraction) and apellet (light membrane fraction). Protein concentrations were measuredusing Protein Assay Kit II from BioRad with bovine serum albumin as thecalibration standard. In some experiments, cell pellets were lysed asabove, but without a freezing step. To test effects of cytochrome c oncaspase activity, some samples were treated with 10 μg/ml bovinecytochrome c (Sigma Chemical, St. Louis, Mo.) throughout the entireisolation procedure. In some experiments, mitochondrial fractions wereprepared from lysed 697-neo and 697-Bcl-2 cells by the rat livermitochondrial methods of Mancini and collaborators (Mancini, et al.,1998) and used without freezing.

Example 3 Immunoblotting

[0098] Subcellular fractions (50 μg protein per lane) were resolved bySDS-PAGE on 12% or 16% gels (Novex, La Jolla, Calif.) and transferred toImmobilon PVDF membranes (Millipore, Bedford, Mass.). Membranes wereblocked in PBS/0.1% Tween 20 (PBST)+0.4 % casein (I-block, Tropix,Bedford, Mass.). Blots were incubated in 1 μg/ml primary antibodydiluted in PBST/casein for 1 hour. Following three washes in PBST, blotswere incubated for one hour in 1:15,000 dilutions of alkalinephosphatase conjugated goat antirabbit IgG or goat anti-mouse IgG(Tropix) in PBST/casein. Blots were then washed twice with PBST, twicein assay buffer (10 mM diethanolamine, pH 10.0, 1 mM MgCl₂) and thenincubated in 250 μM chemiluminescent substrate CSPD (Tropix) in assaybuffer and exposed to Biomax film (Kodak, Rochester, N.Y.) overnight.

[0099] In some cases, following the secondary antibody incubations, theblots were washed with 10 mM Tris, pH9.5, 1 mM MgCl₂. The blots werethen incubated for 30 minutes in 1.25 μg/ml DDAO phosphate (Amersham,Arlington Heights, Ill.) dissolved in the Tris buffer. The blots werescanned using the STORM fluorescence imager (Molecular Dynamics,Sunnyvale, Calif.). The antibodies used were against Bcl-2 (TransductionLabs, Lexington, Ky.), caspase-3 (Srinivasan, et al., 1998), cytochromec (Pharmingen, San Diego, Calif.), cytochrome oxidase, subunit IV(Molecular Probes, Portland, Oreg.), D4-GDP dissociation inhibitor(D4-GDI) (a gift of Dr. G. Bokoch, Scripps Research Institute, La Jolla,Calif.) and poly(ADP-ribose) polymerase (PARP) (Enzyme Systems,Livermore, Calif.).

Example 4 Activity Assays

[0100] Caspase activity was measured by mixing 50 μl of anenzyme-containing fraction and 200 μl of 25 μM DEVD-amc(Asp-Glu-Val-Asp-aminomethylcoumarin) substrate in ICE buffer (20 mMHEPES, 1 mM EDTA, 0.1 % CHAPS, 10 % sucrose, 5 mM DTT, pH 7.5) induplicate Cytoplate wells. Product formation was monitored by theincrease in fluorescence (ex=360 nm, em=460 nm) over 1 -2 hours at 30°C. using the CytoFluor 4000 plate reader (Perseptive Biosystems,Framingham, Mass.). For kinetic studies, the substrate concentration wasvaried in the range 1 -100 μM. For inhibition studies the enzyme waspretreated with 150 μl inhibitor for 30 min at room temperature prior tothe addition of 50 μl of 50 μM substrate solution. Inhibitor IC₅₀ valueswere determined using the equation:

ΔFL/Δt=(ΔFL/Δt)_(o)/(1+[I]/IC ₅₀)

[0101] ΔFL/Δt is the observed initial rate of fluorescence change atinhibitor concentration [I] and (ΔFL/Δt)_(o), is the initial ratefluorescence change for the uninhibited enzyme.

Example 5 Activation Assays

[0102] Heavy membrane samples were diluted to 1 mg/ml in hypotonicbuffer or in 0.25 M sucrose, 10 mM MOPS, 2 mM EDTA, pH 7.4 (Mancini, etal., 1998) containing 5 mM DTT with or without 1% NP40. Caspaseactivation was induced by adding either 60-160 ng/ml recombinant murinecaspase-1 (in bacterial lysate), 2 μg/ml of purified human granzyme B(Enzyme Systems Products, Livermore, Calif.) or buffer, and incubatingthe samples for 60 min at 30° C. or 37° C. After the activation period,the heavy membrane pellet was removed from the sample by centrifugationfor 10 min at 14,000×g at 4° C. The DEVD-amc cleaving activities in theresulting supernatants were corrected for the activity of the exogenousenzymes. To examine the time course of spontaneous activation of caspaseactivity from membranes, 50 μl of heavy membrane slurry containing50-100 μg total protein was mixed with 200 μl hypotonic buffercontaining 25 μM DEVD-amc substrate and 6 mM DTT in 96 well Cytofluorplates and fluorescence was measured over time. At selected time points,aliquots were removed from some wells, centrifuged for 10 min at14,000×g to remove the heavy membranes and the supernatant was addedback into the 96 well plate to measure the soluble DEVD-amc cleavageactivity. In some experiments, subcellular fractions were treated with 1μg/ml bovine cytochrome c (Sigma) and 50 μM dATP (New England Biolabs,Beverly, Mass.) (final concentrations) for 40 min at 30° C. prior tomeasurement of caspase activity.

Example 6 Recombinant Capase Production

[0103] BL21 (DE3) cells harboring a plasmid containing the cloned humancaspase-3 cDNA (Femandes-Alnemri, et al., 1994) (provided by Dr. E.Alnemri, Thomas Jefferson University) was ligated into the Bam HI/Xho Isites of pET21b (Novagen, Madison, Wis.) and were grown in one liter LBmedium containing 0.1 mg/ml ampicillin at 37° C. When the culturedensity reached A₆₀₀=1, IPTG (Sigma) was added to a concentration of 1mM and the culture was incubated at 25° C. for three hours. The cellswere harvested by centrifugation at 2,000×g for 15 min at 4° C. Thecells were lysed using one freeze-thaw cycle in 100 ml Binding buffer(20 mM TrisCl, 500 mM NaCl, 5 mM imidazole, 0.1% triton X-100) with 0.1mg/ml lysozyme. Cell debris was removed from the sample bycentrifugation at 20,000×g, for 30 min at 4° C. The lysed cells weretreated just prior to centrifugation with MgCl₂ and DNase I to reduceviscosity. The supernatant was filtered through a 0.45 μm syringe filterand loaded onto a 1 ml Ni+² -charged HiTrap Chelating column (AmershamPharmacia, Uppsala, Sweden) at a 1 ml/min flow rate. The column waswashed at 1 ml/min with 10 ml Binding buffer followed by 10 ml BindingBuffer containing 60 mM imidazole. The caspase-3 protein was eluted fromthe column using a 30 ml linear gradient of imidazole (60-500 mM).

[0104] Recombinant murine caspase-1 was expressed using BL21 (DE3) pLysS cells harboring pET3ap30mICEFLAG plasmid (a generous gift of Drs. H.R.Horvitz and Ding Xue, MIT) which contains the p30 caspase-1 cDNAinserted into the Nde I/BamH I sites of the pET3a expression vector(Novagen). A three liter culture was grown at 37° C. in Induction medium(20 g/l tryptone, 10 g/l yeast extract, 6 g/l NaCl, 3g/l Na₂HPO₄, 1 g/lKH₂PO₄, 1 mM MgCl₂, 0.1 mM CaCl₂, pH 7.4) containing 0.1 mg/mlampicillin and 0.025 mg/ml chloramphenicol. When the culture reached adensity of A₆₀₀₌1.0, IPTG was added to 1 mM and the culture was shakenat 25° C. for 3 hours. The cells were collected by centrifugation at2000×g for 15 min at 4° C. and resuspended in 100 ml cold buffercontaining 25 mM TrisCl, pH 8.0, 25 mM KCl, 0.1 % triton X-100, and 0.1mg/ml lysozyme (InovaTech, Abbottsford, B.C., Canada). The cells werelysed using one freeze/thaw cycle and lysate was clarified by treatingthe sample with 0.02 mg/ml DNase I, 0.5 mM MgCl₂ (Sigma) for 60 min andthen centrifuging at 20,000×g for 30 min at 4° C. to remove cell debris.

Example 7 In vitro Translation of Caspases

[0105]³⁵S-labeled caspases (wild-type) are obtained by in vitrotranslation in the presence of ³⁵S-methionine using a coupledtranscription/translation system in rabbit reticulocyte lysate using TNTKit (Promega) according to the manufacturer's recommendations.

Example 8 Characterization of Subcellular Fractions

[0106] Subcellular fractions were prepared from 697 cells stablyinfected with retroviral constructs expressing either Bcl-2 cDNA or aneomycin resistance gene (697-Bcl-2 and 697-neo cells, respectively)(Miyashita and Reed, 1993). Nuclear, heavy membrane, light membrane, andcytosolic fractions were isolated from these cells, and characterized byWestern blot analysis with antibodies specific for proteins withdistinct known subcellular distributions, as in Example 3. Antibodiesused were directed against cytochrome oxidase, specific formitochondrial inner membrane (Tzagoloff, 1982), poly(ADP-ribose)polymerase (PARP), specific for nuclei (Berger, 1985), D4-GDPdissociation inhibitor (D4-GDI), specific for cytoplasm (a, et al.,1996) and Bcl-2. The immunoblots were visualized on film bychemiluminescence, except the cytochrome oxidase immunoblot which wasvisualized by chemifluorescence.

[0107] As shown in FIG. 1, the mitochondrial marker was found almostexclusively in the heavy membrane fraction, the nuclear marker only inthe nuclear fraction, and the cytoplasmic marker only in the cytoplasmicfraction. Thus, the fractionation methods employed generated fractionswith the expected subcellular distribution of marker proteins.Importantly, cytoplasmic contamination of the nuclear and membranefractions could not be detected, and only minimal mitochondrialcontamination of nuclear fractions was detected (the diffuse D4-GDIreactive band in the nuclear fraction shown in FIG. 1 is non-specific).Western analysis of fractions from 697-neo cells with an antibody tohuman Bcl-2 (FIG. 1) demonstrated strong reactivity in nuclear and heavymembrane fractions, weaker reactivity in the light membrane fraction,and undetectable signal in cytoplasm, in accord with previous results(Krajewski, et al., 1993; Yang, et al., 1995; Lithgow, et al., 1994).Similar analysis of fractions from 697-Bcl-2 cells showed significantoverexpression.

Example 9 Subcellular Distribution of Cleavage Activity

[0108] Preliminary experiments indicated that caspase activity wasassociated with membranes derived from unstimulated cells. To determinethe subcellular distribution of such caspases, caspase activity in thesubcellular fractions from 697-neo cells was quantitated by incubatingthem with the substrate DEVD-amc, and measuring the increase influorescence over the subsequent 2 hours. DEVD-amc is a useful substratefor all caspases characterized to date, with the exception of caspase-2(Talanian et al., 1997; and data not shown). While most of the DEVD-amccleavage activity (˜75%) was in the cytoplasmic fraction, a substantialamount of the cleavage activity was found in the nuclear, heavy membraneand light membrane fractions (FIGS. 2A and 2C). DEVD-amc cleavageactivity in subcellular fractions of 697 cells transfected with neocontrol or Bcl-2 expression vectors were fractionated and the caspaseactivity of each subcellular fraction was assayed. The observed cleavageactivity values in the histogram are normalized for constant number ofcells (FIG. 2A-2B) or mg protein (FIG. 2C-2D). The values listed foreach column in A and B indicate the percent of total cleavage activitypresent in each fraction. The error bars in FIGS. 2A-2D indicate therange of observed values for two independent 697 cell preparations.

[0109] The major DEVD-amc cleaving activity in each fraction was indeedcaspase activity since it was potently blocked by specific caspaseinhibitors (Table I, column 1 Example 13, and data not shown).

Example 10 Bcl-2 Suppression of Membrane-Derived Caspase Activity

[0110] We next examined the effect of Bcl-2 on the caspase activities inthe various subcellular fractions. When subcellular fractions derivedfrom 697-Bcl-2 cells were prepared and incubated with DEVD-amcsubstrate, substantially reduced caspase activity was observed in thenuclear and heavy membrane fractions compared with 697-neo cells (FIG.2B). This Bcl-2 effect was evident when the caspase activity wasmeasured on a per cell basis or per mg protein and resulted in an 80-90%reduction in caspase activity in these fractions (FIGS. 2B and 2D). Theeffect of Bcl-2 expression on caspase activity in these fractions wasspecific, since little if any suppression was seen in the activitiesobserved in the cytoplasmic or light membrane fractions (FIGS. 2A-2D).These observations suggested that the membrane-associated caspaseactivity was not simply derived from a small percentage of apoptoticcells in the 697-neo cultures whose numbers were suppressed in the697-Bcl-2 cultures. If that were the case, one would also have expect tosee major differences in caspase activities between cytoplasmicfractions derived from 697-neo vs. 697-Bcl-2 cells. Indeed, controlexperiments demonstrated that when 697-neo cells were induced to undergoapoptosis by staurosporine treatment, the major increase in caspaseactivity was found in the cytoplasm (data not shown).

[0111] The ability of Bcl-2 to suppress membrane-associated caspaseactivity was not limited to the 697 lymphoblastoid cells, since similareffects were observed in Jurkat T cells and FL5.12 cells (data notshown). Since the present data, as well as other published studies, havedemonstrated that Bcl-2 protein is found predominantly in nuclearenvelope and heavy membrane fractions (FIG. 1; Krajewski et al., 1993;Yang et al., 1995), the present results were compatible with thepossibility that Bcl-2 might act locally to regulate thismembrane-derived caspase activity. In an effort to begin analyzing suchmechanisms, we further characterized this membrane-derived,Bcl-2-suppressible caspase activity and focused our efforts on the heavymembrane fraction.

Example 11 Spontaneous Activation and Membrane Release ofMembrane-Derived Caspade Activity

[0112] It was possible that the membrane associated caspase activity wasdue either to an active membrane-bound enzyme, or alternatively, to thespontaneous activation and release of a soluble active enzyme. Thereforea set of experiments was designed to distinguish between these twopossibilities. First, to freshly prepared heavy membranes derived from697-neo cells (neo-membranes), hypotonic buffer and DEVD-amc substrateat room temperature was immediately added, and the emergence of amcfluorescence over a 90 minute period (FIG. 3A) was measured. TheDEVD-amc cleavage activity of was measured by adding 20 μg of freshlyprepared membranes into hypotonic buffer containing 20 μM DEVD-amc(final concentration). The evolution of amc product was measured by thechange in fluorescence (ex=360 nm, em=460 nm) at room temperature. Thedata demonstrate that there is little detectable fluorescence changeover the first 15 minutes of incubation, but after this lag period, therate of amc production increases markedly (FIG. 3A). These resultsindicated that the freshly prepared membranes did not contain activecaspase, but that activation occurred spontaneously during theincubation period. To assess whether this newly activated caspase wassoluble or membrane bound, membranes were incubated for differentperiods of time (0 to 90 minutes), following which the samples werecentrifuged for 10 minutes at 14,000×g at 10° C. and the resultingsupernatants were assayed for caspase activity with DEVD-amc substrate.These data demonstrated that very little caspase activity was present inthe supernatant initially, but that soluble caspase activity appearedthereafter (FIG. 3B). Quantitative analysis of these data demonstratedthat for each supernatant, fluorescence increased linearly, indicatingthat once released from the membranes, no further activation occurred.Furthermore, the slopes of these curves (FIG. 3B) approximate theinstantaneous slopes of the corresponding time points in the progresscurve for the heavy membrane slurry (FIG. 3A). Therefore, all of thecaspase-3 activity can be accounted for in the supernatant fraction,indicating that all active enzyme had been released from the membranes.In contrast to the neo-membranes, membranes derived from the 697-Bcl-2cells (Bcl-2-membranes) failed to generate significant DEVD-amc cleavingactivity (FIG. 3A).

Example 12 Procaspase-3 Presence in Heavy Membranes

[0113] The lack of DEVD-amc cleaving activity in the Bcl-2-membranescould be due either to the absence of activatable procaspase orsuppression of procaspase activation. To distinguish between thesealternatives, first, Western blot analysis was performed on the membraneand cytosolic fractions with antibodies specific for caspase-3 (Example3), since the measured DEVD-amc cleavage activity is in fact due tocaspase-3 (see below). The results (FIG. 4) demonstrate the presence ofa caspase-3 reactive band that is of similar intensity in both theneo-membranes and Bcl-2-membranes, and that is approximately the sizeexpected for the procaspase zymogen. Interestingly, the electrophoreticmobility of the membrane-derived bands was slightly slower than that ofcytoplasmic procaspase-3.

[0114] To further demonstrate the presence of procaspase-3 in both neo-and Bcl-2-membranes, we attempted to activate these fractions bytreatment with exogenous caspase-1, since procaspases can be activatedby proteolytic cleavage at aspartic acid residues between their largeand small subunits (Srinivasula et al., Proc. Natl. Acad Sci. USA93:14486-14491, 1996; Stennicke and Salvesen, J. Biol. Chem.272:25719-25723, 1997; Salvesen and Dixit, Cell 91:443-446, 1997). As wehave shown above, membranes derived from Bcl-2 cells showed almost nocaspase activity when measured under our standard conditions. However,treatment of the Bcl-2-membranes with caspase-1 caused a robustinduction of enzymatic activity (FIG. 5). In this experiment, heavymembrane fractions (containing 50 μg total protein) from 697-Bcl-2 and697-neo cells were re-suspended and treated with murine caspase-1 forone hour at room temperature. Following centrifugation, the DEVD-amccleavage activity of the resulting supernatant was measured. TheDEVD-amc cleavage activity of caspase-1 treated samples was correctedfor exogenous caspase-1 activity by subtracting the fluorescence ofcontrol samples containing only caspase-1 from the observedfluorescence. The error bars in FIG. 5 represent the standard deviationof the observed values in 3 independent experiments. The neo-membraneswere also activated by exogenous caspase-1. But importantly, followingactivation, the resulting caspase activities from the Bcl-2- andneo-membranes were always similar, within a factor of two (FIG. 5).Together with the procaspase-3 immunoblot data, this supports theconclusion that comparable levels of procaspase-3 are present in neo-and Bcl-2-membranes.

[0115] Caspase-1 treatment of membranes not only activated theendogenous caspase activity, but also released it from the membranes,since the activity remained in the supernatant when the membranes wereremoved by centrifugation (FIG. 5). This induction and release were dueto the proteolytic activity of caspase-1, since the caspase-1 activationcould be completely blocked by 200 nM acYVAD-aldehyde which inhibitscaspase-1, but not the membrane caspase, at this concentration (data notshown). These results indicate that both neo- and Bcl-2-expressing cellscontain similar amounts of a membrane-associated inactive procaspasethat can be activated by caspase-1. However, without exogenous caspasetreatment, only membranes derived from the neo-expressing cellsdemonstrated spontaneous caspase activation.

Example 13 Characterization of Induced and Spontaneous CaspaseActivities

[0116] The membrane-derived caspase activities were furthercharacterized by measuring the inhibition of DEVD-amc cleavage byseveral peptide aldehyde inhibitors (Table I). The IC₅₀ values for theinhibition of DEVD-amc activity derived from activated Bcl-2-membranesare quite similar to those for the inhibition of the activity derivedfrom neo-membranes, suggesting that caspase-1 activates the sameprocaspase in both membrane preparations. Furthermore, these IC₅₀ valuesare similar to those for the spontaneously activated DEVD-amc activityderived from neo-membranes, suggesting that the spontaneous andcaspase-1-induced activities derive from the same caspase. In all cases,the inhibition data fit well to a simple competitive inhibition curve,suggesting that each DEVD-amc activity arose from a single caspaserather than a mixture of enzymes. The observed IC₅₀ values for themembrane associated caspases are very similar to those for purifiedfully-processed recombinant human caspase-3. Kinetic measurements alsoindicate that K_(m) values for hydrolysis of DEVD-amc by themembrane-derived caspases (10 μM) are similar to that observed withfully processed caspase-3 (Nicholson et al., Nature 376:3743, 1995).N-terminal microsequence analysis of activated, affinity purified heavymembrane caspase confirms that this enzyme is indeed human caspase-3.TABLE I Heavy membrane (HM) derived caspases from various cell types andrecombinant human caspase-3: Inbibition by peptide aldehydes. IC₅₀ (nM)697-neo HM 697-neo HM 697 Bcl-2 HM cortical cell HM MN9D HM (spontaneous(caspase-1 (caspase-1 (caspase-1 (caspase-1 r-caspase-3 inhibitoractivity) treated) treated) treated) treated) (His)₆ DEVD-ald 2.3 2.81.3 1.0  0.72 1.0 DFLD-ald 3.4 4.5 3.6 2.3 2.5 1.5YVAD-ald >10,000 >10,000 >10,000 >10,000 >10,000 >10,000

[0117] To determine if the presence of membrane-associated caspaseactivity is a general property of mammalian cells, the DEVD-amc cleavageactivity in heavy membranes from two other cell sources was measured:mouse E 15 primary brain cortical cells and the mouse dopaminergic MN9Dcell line (Choi et al., Neurobiology 89:8943-8947, 1992). Heavy membranefractions were prepared using identical procedures to those used for the697 cells and were activated with caspase-1. These fractions contained amembrane-associated caspase activity with similar cleavage activitiesper mg protein as observed in 697 cells (data not shown) and that wasblocked by caspase inhibitors with a similar potency to that observedwith fractions derived from 697 cells or with recombinant caspase-3(Table I). Accordingly, the existence of membrane-derived caspaseactivity is not specific to 697 cells, but appears to be a more generalphenomenon.

Example 14 Exogenous Cytochrome C and Membrane Associated Procaspase-3

[0118] Several recent reports have shown that the release of cytochromec from mitochondria can cause the activation of cytoplasmic caspase-3(Liu et al., Cell 86:147-157, 1996; Li et al., Cell 91:479-489, 1997).Other reports have demonstrated that cytochrome c is released frommitochondria following apoptotic insults and that Bcl-2 can inhibit thatrelease (Kluck et al., Science 275:1132-1136, 1997; Yang et al., Science275:1129-1132, 1997). Thus, it was possible that the difference observedbetween caspase activities in heavy membranes from Bcl-2- andneo-expressing cells simply reflected inhibition by Bcl-2 of cytochromec release during preparation of the heavy membrane fractions or duringsubsequent incubation of these fractions. To investigate thispossibility, cell fractionation was performed in the presence ofexogenous cytochrome c and measured whether this influenced caspaseactivation. If the Bcl-2-membranes had low caspase activity because of aBcl-2 effect on cytochrome c sequestration, then the addition ofexogenous cytochrome c during membrane fractionation should increase thecaspase activity derived from those membranes to the levels seen inmembranes from neo-cells. Accordingly, during the fractionationprocedure for heavy membranes from neo- and Bcl-2-expressing cells,following Dounce homogenization, the sample was split into twofractions. One fraction was processed with standard buffers, while tothe other fraction 10 μg/ml of bovine cytochrome c was added, and 10μg/ml to the buffers used to suspend and wash the heavy membranes. Thisconcentration of cytochrome c was chosen since it represents theestimated total amount of cytochrome c present in the starting cellpellets (Li et al., J. Biol. Chem. 272:30299-30305, 1997). Finally,these membranes were resuspended in 1 μg/ml cytochrome c plus 50 μMdATP, incubated, and then assayed for DEVD-amc cleaving activity.Aliquots of the cytochrome c-treated heavy membranes and cytoplasmicfractions were then incubated with hypotonic buffer containing 50 μMdATP/1 μg/ml cytochrome c for 40 min at 30° C., while the membranes andcytoplasmic samples that had not been treated with cytochrome c wereincubated only with buffer. Each sample was then centrifuged, andDEVD-amc cleavage activity in the supernatant was measured. The data inFIG. 6 represents three equivalent experiments (FIG. 6A). This activitywas compared to that from our usual membrane preparations preparedwithout cytochrome c, and incubated without cytochrome c or DATP.

[0119] The data demonstrate that inclusion of cytochrome c duringmembrane fractionation and incubation has no effect on membrane-derivedcaspase activity; the activity in the membranes derived fromBcl-2-expressing cells remained low compared to the activity in theneo-membranes, and furthermore, there was also no effect of cytochrome con the caspase activity derived from the neo-membranes (FIG. 6A).Although the cytochrome c treatments did not activate themembrane-associated caspase, the enzyme could still be activated bysubsequent treatment with exogenous caspase-1 (data not shown). The lackof a cytochrome c effect on the activation of the membrane caspase wasnot due to an inactive preparation of cytochrome c, since the DEVD-amccleavage activity of the cytoplasmic fractions from both neo and Bcl-2cells were strongly activated by inclusion of cytochrome c duringfractionation and assay (FIG. 6B). Therefore, Bcl-2 expressionsuppresses the activation of the membrane-associated procaspase-3, butthis effect is not overcome by addition of exogenous cytochrome c.Furthermore, Bcl-2 overexpression did not affect the ability ofcytochrome c to activate caspase-3 in cytoplasmic fractions.

Example 15 Release of Membrane-Derived Caspase is not via Simple LeakageFrom Organelles

[0120] A recent report described the presence of procaspase-3 in theintermembrane space within mitochondria (Mancini et al., J. Cell Biol.140:1485-1495 1998). Thus, it was possible that this material couldaccount for the activatable caspase activity that was measured in themitochondria-containing heavy membrane fractions. Furthermore, it waspossible that the spontaneous activity that was measured in membranefractions from 697-neo cells was due to leakage of active caspase frommitochondria, and that mitochondria isolated from 697-Bcl-2 cells weresimply less leaky (Yang et al., Science 275:1129-1132, 1997). However,several experiments suggested that the activity measured was not due toleakage from mitochondria, and that the activity is distinct from thatdescribed by Mancini et al., supra.

[0121] First, whether the addition of 1% NP40 to neo-membranes affectedthe level of either spontaneous activity or the activity induced bycaspase-1 or granzyme B was tested. It was reasoned that if procaspaseand/or active caspase was sequestered within organelles, then enhancedactivity would be measured in the presence of NP-40. Treatment with 1%NP-40 was sufficient to release almost all of the cytochrome c presentin heavy membrane preparations (data not shown). Furthermore, it wasshown by Mancini and colleagues that treatment of their mitochondrialpreparations with 1% NP-40 allowed granzyme B to cleave procaspase-3whereas no cleavage was observed in the absence of detergent (Mancini etal., J. Cell Biol. 140:1485-1495, 1998). However, the present resultsdemonstrate that 1% NP-40 had little effect either on spontaneousactivity or the activity induced by treatment with caspase-1 or granzymeB (FIG. 6A). In this experiment, 160 μl of neo-membranes were dilutedwith 180 μl hypotonic buffer and treated with 40 μl 10% NP-40 detergentor dH₂O (final vol=380 μl). The diluted membranes were activated by theaddition of 20 μl granzyme B or caspase-1 lysate or buffer, andincubated for 60 min at 30 ° C. Following activation, the heavymembranes were removed by centrifugation and the DEVD-amc cleavingactivity of each sample was measured by adding 50 μl of each supernatantto 200 μl of 25 μM DEVD-amc substrate in ICE buffer (FIG. 7A).

[0122] Next, to analyze whether membrane preparations from 697-Bcl-2cells may have low spontaneous activity due to enhanced sequestration ofa caspase, we added DEVD-amc to Bcl-2- and neo-membrane preparations,incubated them in buffer alone or buffer plus 1% NP-40, and measured theappearance of fluorescence. In this experiment, the effect of NP-40 onthe progress curve for heavy membrane catalyzed DEVD-amc hydrolysis wasmeasured by adding 50 μl freshly prepared neo- or Bcl-2-membranes to 200μl 25 μM DEVD-amc in hypotonic buffer pH 7.5 (containing 4 mM DTT) withor without 1% NP-40 detergent. The results indicate that 1% NP-40 hadonly a minor effect on the magnitude or rate of fluorescence increase.Preparations derived from 697-Bcl-2 cells had low activity regardless ofwhether 1% NP-40 was present, demonstrating that this low level ofactivity was not due to sequestration of an active caspase.

[0123] Lastly, mitochondrial fractions from 697-neo and 697-Bcl-2 cellswere prepared using the methods described by Mancini et al. (1998) tomore directly assess the relationship between our results and theirpublished data In this experiment, diluted membranes, with or without 1%NP-40, were activated by the addition of granzyme B or buffer for 60min, centrifuged, and assayed for DEVD-amc cleavage activity asdescribed in FIG. 7A. As shown in FIG. 7C, fractions from both 697-neoand 697-Bcl-2 made by these methods have granzyme B-activatable caspaseactivity in the absence of NP40. However, in the presence of 1% NP40,granzyme B treatment yielded enhanced caspase activity. Thus, underthese conditions, granzyme B generates caspase activity in both NP-40independent and dependent manners.

[0124] From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method for identifying membrane derived caspase activity,comprising incubating a membrane fraction comprising heavy or nuclearmembranes under conditions and for a time sufficient to allow for theevolution of caspase activity, and subsequently detecting caspaseactivity.
 2. The method of claim 1, wherein caspase activity is detectedby measuring caspase substrate turnover.
 3. The method of claim 2,wherein substrate turnover is measured by time course analysis.
 4. Themethod of claim 2, wherein substrate turnover is measured by endpointanalysis.
 5. The method of claim 2, wherein substrate turnover isdetected by a method selected from the group consisting of fluorescencespectroscopy, mass spectrometry, HPLC, colorimetry, fluorography,radiography, gel electrophoresis, chromatography and N-terminal peptidesequencing.
 6. The method of claim 1, wherein caspase activity isdetected by determining caspase processing said processing providinglarge and small caspase subunits.
 7. The method of claim 6, whereinlarge and small caspase subunits are detected by a method selected fromthe group consisting of fluorescence spectroscopy, mass spectrometry,HPLC, colorimetry, fluorography, radiography, gel electrophoresis,chromatography and N-terminal peptide sequencing.
 8. The method of anyone of claims 1-7, wherein the membrane fraction is derived fromnon-apoptotic cells.
 9. The method of any one of claims 1-7, wherein themembrane fraction is derived from cells treated with a stimulator ofapoptosis.
 10. The method of claim 9, wherein the stimulator ofapoptosis is selected from the group consisting of deprivation of agrowth factor, staurosporine, anti-fas antibody, ultravioletirradiation, gamma irradiation, and Tumor Necrosis Factor.
 11. A methodfor identifying an inhibitor of the activity of a membrane derivedcaspase, comprising contacting a membrane fraction with a caspasesubstrate in the presence and absence of at least one candidateinhibitor; and comparing the levels of caspase substrate turnover, andtherefrom identifying an inhibitor of the activity of a membrane derivedcaspase.
 12. The method of claim 11, wherein the caspase substratecomprises a site cleaved by a caspase selected from the group consistingof a protein, a polypeptide, an oligopeptide, a peptide mimetic and apeptide.
 13. The method of claim 12, wherein the substrate comprises thepeptide DEVD.
 14. The method of claim 11, wherein the membrane fractionis prepared from un-stimulated tissue culture cells selected from thegroup consisting of 697 lymphoblastoid cells, E15 primary brain corticalcells, MN9D cells, Jurkat T cells, and FL5.12 cells.
 15. The method ofclaim 11, wherein the membrane fraction comprises membranes selectedfrom the group consisting of heavy membranes and nuclear membranes. 16.The method of claim 11, wherein the membrane fraction comprises heavymembranes.
 17. The method of claim 11, wherein substrate turnover isdetected by time course analysis.
 18. The method of claim 11, whereinsubstrate turnover is detected by endpoint analysis.
 19. The method ofclaim 17 or 18, wherein caspase substrate turnover detection isperformed by a method selected from the group consisting of fluorescencespectroscopy, mass spectrometry, HPLC, colorimetry, fluorography,radiography, gel electrophoresis, chromatography and N-terminal peptidesequencing.
 20. The method of claim 11, wherein the membrane fraction isderived from cells expressing pro-apoptotic polypeptides.
 21. The methodof claim 11, further comprising incubating the membrane fraction with acaspase activator prior to or concurrent with the addition of thecaspase substrate.
 22. The method of claim 11, wherein the membranefraction is derived from non-apoptotic cells.
 23. The method of claim11, wherein the membrane fraction is derived from cells treated with astimulator of apoptosis.
 24. The method of claim 23, wherein thestimulator of apoptosis is selected from the group consisting ofdeprivation of a growth factor, staurosporine, anti-fas antibody,ultraviolet irradiation, gamma irradiation and Tumor Necrosis Factor.25. A method for identifying an enhancer of the activity of a membranederived caspase, comprising contacting a membrane fraction with acaspase substrate in the presence and absence of at least one candidateenhancer; and comparing the levels of caspase substrate turnover, andtherefrom identifying an enhancer of the activity of a membrane derivedcaspase.
 26. The method of claim 25, wherein the caspase substratecomprises a site cleaved by a caspase selected from the group consistingof a protein, a polypeptide, an oligopeptide, a peptide mimetic, and apeptide.
 27. The method of claim 26, wherein the substrate comprises thepeptide DEVD.
 28. The method of claim 25, wherein the membrane fractionis prepared from un-stimulated tissue culture cells selected from thegroup consisting of 697 lymphoblastoid cells, E15 primary brain corticalcells, MN9D cells, Jurkat T cells and FL5.12 cells.
 29. The method ofclaim 25, wherein the membrane fraction comprises membranes selectedfrom the group consisting of heavy membranes and nuclear membranes. 30.The method of claim 25, wherein the membrane fraction comprises heavymembranes.
 31. The method of claim 25, wherein substrate turnover isdetected by time course analysis.
 32. The method of claim 25, whereinsubstrate turnover is detected by endpoint analysis.
 33. The method ofclaim 31 or 32, wherein caspase substrate turnover detection isperformed by a method selected from the group consisting of fluorescencespectroscopy, mass spectrometry, HPLC, colorimetry, fluorography,radiography, gel electrophoresis, chromatography and N-terminal peptidesequencing.
 34. The method of claim 25, wherein the membrane fraction isderived from cells expressing an anti-apoptotic polypeptide.
 35. Themethod of claim 34, wherein the anti-apoptotic polypeptide is Bcl-2. 36.The method of claim 34, further comprising incubating the membranefraction with a caspase activator prior to or concurrent with theaddition of the caspase substrate.
 37. The method of claim 25, whereinthe membrane fraction is derived from non-apoptotic cells.
 38. Themethod of claim 25, wherein the membrane fraction is derived from cellstreated with a stimulator of apoptosis.
 39. The method of claim 38,wherein the stimulator of apoptosis is selected from the groupconsisting of deprivation of a growth factor, staurosporine, anti-fasantibody, ultraviolet irradiation, gamma irradiation and Tumor NecrosisFactor.
 40. The method of claim 25, wherein an exogenous anti-apoptoticpolypeptide is added prior to or concurrently with the addition of thecaspase substrate.
 41. A method for identifying an inhibitor or enhancerof the evolution of caspase processing within a membrane fraction,comprising contacting a membrane fraction with at least one candidateinhibitor or candidate enhancer; and detecting the presence of large andsmall caspase subunits, and therefrom determining the level of caspaseprocessing, wherein a decrease in processing indicates the presence of acaspase processing inhibitor, and wherein an increase in processingindicates the presence of a caspase processing enhancer.
 42. The methodof claim 41, wherein the membrane fraction is prepared fromun-stimulated tissue culture cells selected from the group consisting of697 lymphoblastoid cells, E15 primary brain cortical cells, MN9D cells,Jurkat T cells, and FL5.12 cells.
 43. The method of claim 41, whereinthe membrane fraction comprises membranes selected from the groupconsisting of heavy membranes and nuclear membranes.
 44. The method ofclaim 41, wherein the membrane fraction comprises heavy membranes. 45.The method of claim 41, wherein large and small caspase subunits aredetected by a method selected from the group consisting of fluorescencespectroscopy, mass spectrometry, HPLC, colorimetry, fluorography,radiography, gel electrophoresis, chromatography and N-terminal peptidesequencing.
 46. The method of claim 41, wherein the membrane fraction isderived from cells expressing anti-apoptotic polypeptides.
 47. Themethod of claim 46, wherein the anti-apoptotic polypeptide is Bcl-2. 48.The method of claim 41, wherein the membrane fraction is derived fromnon-apoptotic cells.
 49. The method of claim 41, wherein the membranefraction is derived from cells treated with a stimulator of apoptosis.50. The method of claim 49, wherein the stimulator of apoptosis isselected from the group consisting of deprivation of a growth factor,staurosporine, anti-fas antibody, ultraviolet irradiation, gammairradiation and Tumor Necrosis Factor.
 51. A method of identifying acompound that modulates membrane fraction derived caspase activity,comprising incubating a membrane fraction, an inhibitor of apoptosis,and a caspase substrate in the presence and absence of at least onecandidate compound under conditions and for a time sufficient to allowfor the evolution of caspase activity; and comparing the levels ofcaspase substrate turnover, thereby identifying a compound thatmodulates membrane derived caspase activity.
 52. The method of claim 51,wherein the caspase substrate comprises a site cleaved by a caspase andis selected from the group consisting of a protein, a polypeptide, anoligopeptide, a peptide mimetic, and a peptide.
 53. The method of claim52, wherein the substrate comprises the peptide DEVD-amc.
 54. The methodof claim 51, wherein the membrane fraction is prepared fromun-stimulated tissue culture cells selected from the group consisting of697 lymphoblastoid cells, E15 primary brain cortical cells, MN9D cells,Jurkat T cells and FL5.12 cells.
 55. The method of claim 51, wherein themembrane fraction comprises membranes selected from the group consistingof heavy membranes and nuclear membranes.
 56. The method of claim 51,wherein the membrane fraction comprises heavy membranes.
 57. The methodof claim 51, wherein substrate turnover is detected by time courseanalysis.
 58. The method of claim 51, wherein substrate turnover isdetected by endpoint analysis.
 59. The method of claim 57 or 58, whereincaspase substrate turnover detection is performed by a method selectedfrom the group consisting of fluorescence spectroscopy, massspectrometry, HPLC, colorimetry, fluorography, radiography, gelelectrophoresis, chromatography and N-terminal peptide sequencing. 60.The method of claim 51, wherein the membrane fraction contains theinhibitor of apoptosis.
 61. The method of claim 60, wherein the membranefraction is derived from cells expressing Bcl-2.
 62. The method of claim51, wherein the inhibitor of apoptosis is a Bcl-2 polypeptide or afunctional fragment thereof.
 63. The method of claim 51, furthercomprising incubating the membrane fraction with a caspase activatorprior to or concurrent with the addition of the caspase substrate. 64.The method of claim 51, wherein the membrane fraction is derived fromnon-apoptotic cells.
 65. The method of claim 51, wherein the membranefraction is derived from cells treated with a stimulator of apoptosis.66. The method of claim 65, wherein the stimulator of apoptosis isselected from the group consisting of deprivation of a growth factor,staurosporine, anti-fas antibody, ultraviolet irradiation, gammairradiation and Tumor Necrosis Factor.
 67. An inhibitor of the activityof a membrane derived caspase identified by any one of methods 11 and41.
 68. An enhancer of the activity of a membrane derived caspaseidentified by any one of methods 25 and 41.